Polyquarternium-1 synthesis methods

ABSTRACT

A method of making antimicrobial quaternary ammonium polymers, comprising: a) mixing 1,4-bis-dimethylamino-2-butene, water, a first portion of triethanolamine and a first portion of acid; b) adding a 1,4-dihalo-2-butene and heating the reaction mixture; c) adding a second portion of triethanolamine and a second portion of acid, and d) isolating a quaternary ammonium polymer having a molecular weight of at least 26 k.

RELATED CASES

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/609,422, entitled Polyquaternium-1 SynthesisMethods, filed Apr. 17, 2007, now U.S. Pat. No. 7,705,112 which isincorporated herein in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present embodiments relate to an improved method of synthesizinghigh molecular weight polyquaternium-1 and related molecules for use asantimicrobial agents in contact lens solutions.

2. Description of the Related Art

Quaternary ammonium polymers in which the ammonium moieties are part ofthe linear polymeric chains have been used as antimicrobial agents inseveral industries. Polyquaternium-1 (PQ1) is a polymeric quaternaryammonium anti-microbial agent that has been used, for example, inpreserving ophthalmic compositions and disinfecting contact lenses. PQ1is effective against bacteria, algae and fungi. Its chemical name ispoly[(dimethyliminio)-2-butene-1,4-diyl chloride],α-[4-[tris(2-hydroxyethyl)ammonio]-2-butenyl]-ω-[tris(2-hydroxyethyl)ammonio]-dichloride.

U.S. Pat. No. 3,931,319, which is hereby incorporated in its entirety byreference, describes a two-step method for PQ1 synthesis which requiresa high reaction temperature. This leads to significant degradation ofthe target molecule into impurities from which the desired PQ1 isdifficult to separate.

U.S. Pat. No. 4,027,020, which is hereby incorporated in its entirety byreference, describes a procedure for polyquaternium-1 synthesis whichresults in less degradation of the resulting PQ1 than the methoddescribed in U.S. Pat. No. 3,931,319 but still produces a rather lowyield. The procedure disclosed in U.S. Pat. No. 4,027,020 entails mixing1,4-bis-dimethylamino-2-butene with triethanolamine (TEA), the molarratio of the 1,4-bis-dimethylamino-2-butene to the TEA amine being from2:1 to 30:1 followed by the addition of 1,4-dichloro-butene to themixture in a molar amount equal to the sum of the molar amount of the1,4-bis-dimethylamino-2-butene plus one-half the molar amount of TEA.The reaction time is 1-10 hours.

A major weakness of the method taught in U.S. Pat. No. 4,027,020 is thatthe TEA end-capping efficiency is low. As such, the final productcontains a significant amount of polymers with no end caps or polymersend-capped with groups other than TEA. These malformed polymers aredifficult to separate from polyquaternium-1 because of the similarity inthe main chain of the polymeric molecules. Degraded or malformedpolymers of PQ1 have reduced anti-bacterial efficacy and cannotsubstitute for PQ1 in clinical use.

Soft contact lenses usually attract and accumulate quaternary ammoniumantimicrobial agents during the lens cleaning/disinfecting/storingcycles. The accumulated antimicrobial agents in the lens are subsequencereleased once the lens is put in to the eye, causing the contact lenswearer's eye irritation. An effective way to reduce the antimicrobialagents lens uptake is to use low concentration antimicrobial agents.This requires that the antimicrobial agents are of high efficacy.Another way to reduce the eye irritation is to use less cytotoxicantimicrobial agents in MPS.

SUMMARY OF THE INVENTION

One object of the invention is to provide a method of makingantimicrobial quaternary ammonium polymers, comprising:

-   -   a) mixing 1,4-bis-dimethylamino-2-butene, water, a first portion        of triethanolamine and a first portion of acid;    -   b) adding a 1,4-dihalo-2-butene and heating the reaction        mixture;    -   c) adding a second portion of triethanolamine and a second        portion of acid, and    -   d) isolating a quaternary ammonium polymer having an average        molecular weight determined using the proton NMR method of at        least 26 k, preferably greater than 26K or at least 28 k.

Another object of the invention is to provide a method of makingPolyquaternium-1, comprising:

-   a) mixing 1,4-bis-dimethylamino-2-butene, water, a first portion of    triethanolamine and a first portion of acid;-   b) introducing a 1,4-dihalo-2-butene and heating the reaction    mixture;-   c) adding a second portion of triethanolamine and a second portion    of acid, and-   d) isolating Polyquaternium-1 having an average molecular weight    determined using the proton NMR method of 26 k or more, preferably    greater than 26 k or at least 28 k, at a yield of at least about 50%

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows GPC chromatograms for the products of comparative example 1having no acid added to the reaction mixture.

FIG. 2 shows a UV absorbance spectrum of the large PQ1 moleculesaccording to comparative example 1 (without acid added) with peaks atretention times 6.3 minutes and 9.5 minutes.

FIG. 3 shows the GPC chromatograms for the products of example 1 havingacid added.

FIG. 4 shows the UV absorbance spectrum of the synthesized crude productin example 1 at 6 hours reaction time, with peaks at retention times 6.3and 9.5 minutes.

FIG. 5 shows the GPC chromatograms for the products of the reaction asdescribed in comparative example 2.

DETAILED DESCRIPTION

Various multipurpose lens care solutions have been developed over theyears to ensure that contact lenses are essentially pathogen and depositfree. These contact lens solutions typically include anti-microbialsubstances as well as cleaning (active against both lipids andproteins), wetting, conditioning, and other agents for the disinfectionand cleaning of contact lenses during storage after wear. So-called,multipurpose solutions (MPS) can disinfect and clean without harming theeye or lens in addition to wetting.

In accordance with the present invention, high molecular weight PQ1 ismore efficacious in killing microorganisms and less toxic to the eyethan low molecular weight PQ-1.

The present embodiments relate to improved methods for the synthesis ofhigh molecular weight quaternary ammonium polymers by addingtriethanolamine (TEA) in two separate stages. The method involvesaddition of acid to the reaction mixture in separate stages as well. Theoverall method prevents impurity generation and the degradation ofsynthetic quaternary ammonium polymers, including PQ1, during thereaction. Recent experiments have shown that past methods ofsynthesizing quaternary ammonium polymers are not as efficient asoriginally thought. This is due in party to the fact that too little TEAwas used in the reaction admixture.

Regardless of the molar ratio of TEA used, PQ1 synthesized byconventional methods described in U.S. Pat. Nos. 4,027,020 and 3,931,319invariably result in significant PQ1 degradation during the reactionprocess. The molecular structure of PQ1 can be expressed as:

The majority of the degraded molecules are:

A) (HOC₂H₄)₃NCH₂CH═CHCH₂(N(CH₃)₂CH₂CH═CHCH₂)_(n-1)N(CH₃)₂ and

B) H₂C═CHCH═CH(N(CH₃)₂CH₂CH═CHCH₂)mN(HOC₂H₄OH)₃.

These degraded molecules are difficult to separate from PQ1, since bothare polymeric quaternary amine-based like PQ1. Degraded or malformedpolymers of PQ1 have reduced anti-bacterial efficacy and cannot besubstituted for PQ1 in clinical use.

Nucleophilic substitution reactions involving alkyl halides are wellknown in the literature. A nucleophilic agent is a Lewis base which candonate an unshared pair of electrons to form a new covalent bond.(HOC₂H₄)₃NH⁺ is a Lewis acid and, therefore, does not normally reactwith ClCH₂CH═CHCH₂(N(CH₃)₂CH₂CH═CHCH₂)_(n)-1N(CH₃)₂CH₂CH═CHCH₂Cl in theend-capping step of the reaction to form PQ1. Therefore, the currentliterature view is that acids should be avoided in the nucleophilicreaction of the present embodiments for fear that acid could convert thenucleophilic agent (HOC₂H₄)₃N into inactive (HOC₂H₄)₃NH⁺ ions. However,contrary to the current literature view, the present embodiments relateto a synthesis wherein the addition of acid to the reaction mixture doesnot prevent the TEA end-capping reaction.

U.S. patent application Ser. No. 11/609,422, describes an acid catalyzedmethod for PQ1 synthesis. In this method all the raw materials, such as1,4-bis-dimethylamino-2-butene (DA), TEA, 1,4-dichloro-2-butene (DCB)and an acid (e.g., HCl), mixed together directly. The following tworeactions start simultaneously:

In the methods of the prior art that do not include the addition of acidto the reaction mixture, when 1,4-bis-dimethylamino-2-butene,triethanolamine and water are mixed, the hydroxide concentration is veryhigh, usually greater than about 10⁻³ M. Since the nucleophilicity ofhydroxide is much stronger than that of TEA and1,4-bis-dimethylamino-2-butene, large amounts of 1,4-dihalo-2-butene areattacked by hydroxide in the prior art methods, resulting inHOCH₂CH═CHCH₂Cl or HOCH₂CH═CHCH₂OH. As discussed below, hydroxide alsocompetes with TEA in the end-capping reaction of PQ1, resulting lowyield and high impurities for PQ1. Therefore, in the present embodimentsthe presence of acid is advantageous in the reaction admixture toprevent PQ1 degradation, improve the reaction yield and reduce productimpurity, regardless of the molar ratio of1,4-bis-dimethylamino-2-butene to triethanolamine.

Significant PQ1 degradation during the synthesis process can beprevented by adding acid to the reaction admixture. Addition of acidgreatly reduces the formation of degraded impurities and increases theyield of PQ1 in the reaction.

In the present method, the molar amount of DCB should be: (1) enough tocap the two ends of the product of Reaction 1, and (2) maintain acertain amount of excess, so that the PQ1 chain-length extension ofReaction 1 is fast enough to avoid hydrolysis of the end cap group—CH₂Cl to —CH₂OH. A portion of the TEA is added after Reaction 1 iscompleted so that Reaction 2 can take place before the end capped —CH₂Clgroups are hydrolyzed.

Since DCB is a very toxic material, the excess amount must beneutralized by TEA by the end of the reaction. This is usually achievedby adding a large excess of TEA to the reaction mixture. However, aproblem arises because that the molecular weight of the resulting PQ1 isnot as high as desired. This is because the existence of large a excessof TEA also accelerates the end group capping Reaction 2, and so theantimicrobial activity of the product is reduced. Whenever a TEAmolecule reacts with the —CH₂Cl group of the product in Reaction 1, thatside of the PQ1 chain stops chain growth.

According to the present invention, if the total amount of TEA isdivided into two parts, so that one part is added before Reaction 1, andthe second part is added 10 minutes to 8 hours after the process begins,the product's molecular weight is much higher than by using a one-stepTEA addition. In this way the hydrolysis reaction of the end cap group—CH₂Cl can be avoided but the excess amount of DCB can still beneutralized.

In general, the synthesis of PQ1 involves a 1,4-dihalo-2-buteneincluding, for example, 1,4-dichloro-2-butene, 1,4-difluoro-2-butene,1,4-dibromo-2-butene, and/or 1,4-diiodo-2-butene. In a preferredembodiment, the 1,4-dihalo-2-butene is 1,4-dichloro-2-butene.

The following examples are provided for illustrative purposes only, andare in no way intended to limit the scope of the present invention.

Comparative Example 1

PQ1 was synthesized as described in U.S. Pat. No. 4,027,020 using areactant admixture of 1,4-bis-dimethylamino-2-butene with TEA in whichthe molar ratio of 1,4-bis-dimethylamino-2-butene to TEA was about 5:1and the molar ratio of 1,4-dichloro-butene to1,4-bis-dimethylamino-2-butene was about 1.1:1. The reaction was carriedout at 65° C. The proton NMR spectra were obtained for the final productafter it was purified with ultrafiltration. The results are summarizedin Table 1, where the peaks at the chemical shift of 6.5 ppm and 3.7 ppmare for vinyl protons in repeating units and allylic protons adjacent tothe nitrogen in the ending group of the PQ1 molecules, respectively.

Table 1 one shows that the TEA end-capping efficiency is low in 6 hoursreaction at which the reaction was believed by the authors of the U.S.Pat. No. 4,027,020 to be complete. The reaction time was then extendedfrom 6 to 10 hours and the results show that the amount of proton in theend cap group of the polymers is still increasing. Therefore, theend-capping reaction for PQ1 synthesis is not completed at 6 hours andis approximately only 71% complete.

TABLE 1 Peak area at 6.5 Peak area at 3.7 Reaction Time ppm Chemicalshift ppm Chemical shift 6 hours 1.000 0.0343 10 hours  1.000 0.0481

The low end-capping efficiency is due to the low amount of TEA in thereactant admixture. The low TEA concentration in the reaction mixtureslows down its kinetic reaction rate withClCH₂CH═CHCH₂(N(CH₃)₂CH₂CH═CHCH₂)_(n)CH₂CH═CHCH₂Cl. Meanwhile, watermolecules and hydroxide ions (OH—) in the solution may compete with TEAto form OHCH₂CH═CHCH₂(N(CH₃)₂CH₂CH═CHCH₂)_(n)CH₂CH═CHCH₂OH.

FIGS. 1 a, 1 b and 1 c represent the GPC chromatograms for PQ1synthesized with admixtures of 1 mole of 1,4-bis-dimethylamino-2-butene,0.9 moles of TEA, and 1.15 moles of 1,4-dichlo-butene at 65° C. at 2, 6and 10 hours respectively. A GPC-HPLC chromatograph was used to tracethe PQ1 molecular size. The experimental conditions were: an aqueoussolution of 0.045 M KH₂PO₄, 0.45% NaCl and 9.1% CH₃CN as a mobile phasein a Phenomenex BioSep-SEC-S 2000 column and an Agilent 1100 Series HPLCsystem equipped with PDA detector. PQ1 molecules have an absorbance peakat 205 nm but do not have an absorbance peak at 228 nm. However, thedegraded molecules have an absorbance maximum at 228 nm. Therefore thedetection wavelengths of 205 nm and 228 nm are used to trace PQ1 and itsdegradated segments, respectively, during the reaction process.

The broad peak shown in FIGS. 1 a, 1 b and 1 c which ranges from 6 to 10minutes retention time represents polymeric molecules of PQ1 and itsdegraded products. The larger the polymeric molecules, the shorter theretention time will be. The water solvent peak locates at about 10minutes. The peaks beyond 10 minutes represent non-polymeric smallmolecules of either the reactants or bi-products.

As can be seen in FIG. 2, the crude PQ1 product synthesized as describedin the U.S. Pat. No. 4,027,020 without adding acid shows absorbance at228 nm. The absorbance peak shifts to a longer retention time withincrease of reaction time from 8.4 min at 2 hours (see FIG. 1 a) to 9minutes at 10 hours (see FIG. 1 c). FIG. 2 further shows that thespectrum of the large PQ1 molecules at retention time of 6.3 minutes hasno absorbance at 228 nm and that the spectrum at 9.5 minutes possess astrong absorbance at 228 nm. Clearly, there are two or more types ofdifferent polymeric quaternary amines generated in the product mixture.The large polymers are close to PQ1 and the small polymers correspond tothe degraded PQ1.

In the present embodiments, any acid can be used in the syntheticmethod. In preferred embodiments, the acid used does not contain astrong nucleophilic group. Preferred acids include HCl, H₂SO₄, and H₃PO₄but the present embodiments are not limited to these acids. Additionalsuitable acids include acetic acid, succinic acid, and citric acid,among others.

Comparative Example 2

FIG. 5 shows the GPC chromatograms for the products of the reaction asdescribed above in Comparative Example 1 except with a specific reactionadmixture of 1 mole of 1,4-bis-dimethylamino-2-butene, 1.2 moles of TEA,and 1.2 moles of 1,4-dichlo-butene at 60° C. for 18 hours. No acid wasadded to the reaction admixture.

The severe degradation during synthesis process is shown with strongabsorption at 228 nm. The long retention time also indicates that PQ1was degraded into smaller molecular size. Another indication of PQ1degradation in the absence of acid is the increase of peak area at theretention time of 10.5 minutes over reaction time from FIGS. 1 and 5.This peak corresponds to non-polymeric small molecules with similarabsorbance spectrum maximum at 225 nm as that of 228 nm for one of thedegraded PQ1 molecules. It is likely that all PQ1 will eventually bedegraded in to the small molecules during reaction or storage if thetime is long enough.

Comparative Example 3

In order to prevent the side end-capping reaction and increase the mainend-capping reaction rate, the amount of TEA in the admixture of thereactants was increased. Table 2 shows the proton NMR spectrum data forPQ1 synthesized at 65° C. with admixture of 1 mole of1,4-bis-dimethylamino-2-butene, 0.9 moles of TEA, and 1.15 moles of1,4-dichlo-butene (the molar ratio of 1,4-bis-dimethylamino-2-butenewith TEA is 1.11:1 instead of 5:1 as in Example 1 above). The far rightcolumn of Table 2 lists the end-capping percentage over the reactiontime. It can be seen that even at the presence of large excess amount ofTEA, the reaction is still not complete until a time of 4 hours. SeeFIGS. 1 and 2.

TABLE 2 Peak area at 6.5 Peak area at 3.7 End-capping Reaction Time ppmChemical shift ppm Chemical shift efficiency 2 hours 1.000 0.108 94.7% 4 hours 1.000 0.114 100% 6 hours 1.000 0.114 100%

Example 1

10.14 grams (71.3 mmoles) of 1,4-bis-dimethylamino-2-butene, 6.4 grams(42.8 mmoles) of TEA, 4.92 ml of 6N HCl (29.5 mmoles), 18.8 grams ofwater and a stir bar were combined in a 100 ml three-mouth flask. Theflask was submerged into an ice water bath. 9.8 grams of (78.4 mmoles)of 1,4-dichloro-2-butene were slowly added drop-wise into the flaskunder constant stirring. The ice-bath was removed after the1,4-dichloro-2-butene was completely added and the flask was submergedin a warm-water bath (25-40° C.) for 20 minutes. The water bath washeated until the temperature inside the flask reached 70° C. Thereaction was stopped after 21 hours by removing the flask from the waterbath. Variations can be made to the procedure by those skilled in theart for larger scale production to release the heat generated at theinitial stage of the reaction before raising the temperature to above60° C.

FIGS. 3 a, 3 b and 3 c are the GPC chromatograms for the aboveadmixtures with HCl added. The peak at 205 nm in each chromatogram showsthe presence of PQ1, while the lack of peak at 228 nm indicates theabsence of degradation products. FIG. 4 is the spectra of thesynthesized crude product at 6 hours reaction time at 6.3 and 9.5minutes retention time, respectively. It can be seen that there is noabsorbance at 228 nm in the whole 10 hours reaction period when the acidis added to the reaction mixture, indicating that no degraded PQ1 hasbeen formed. FIG. 4 further confirms that there is no absorbance peak at228 nm at the whole retention time range of 6-10 minutes. This resultindicates that the addition of the acid effectively prevented theformation of degraded PQ1.

TABLE 3 Summary of Peak Retention Time for the Products Synthesized withand without Acid Peak retention time without Peak retention time withacid acid Reaction Time 205 nm 228 nm 205 nm 228 nm 2 hours 6.7 8.4 6.9no peak 6 hours 7.3 8.9 6.9 no peak 10 hours  7.6 9.0 6.9 no peak

The absorbance at 205 nm is mainly from the molecule back-bonestructure. Table 3 further shows that the polymer molecular sizedistribution measured at 205 nm is stable in the system where the acidis added.

As one of ordinary skill in the art will appreciate, the above crude PQ1products can be purified by removing the excess amount of TEA, the acid,1,4-dichloro-2-butene and other small molecule byproduct/impuritieswhich are shown up at the retention time of >10 minutes in FIGS. 1 and 3using methanol and/or acetone as solvents.

Example 2 Polyquaternium-1 Synthesis Procedure for Sample #2 in Table 4

10.14 grams (71.3 mmoles) of 1,4-bis-dimethylamino-2-butene, 6.4 grams(42.8 mmoles) of TEA, 4.92 ml of 6N HCl (29.5 mmoles), 18.8 grams ofwater and a stir bar were combined in a 100 ml three-mouth flask. Theflask was then submerged into an ice water bath. 9.8 grams (78.4 mmoles)of 1,4-dichloro-2-butene were added (drop-wise) into the flask underconstant stirring. The ice-bath was removed after all of the1,4-dichloro-2-butene was completely added and the flask was submergedinto a warm-water bath (25-40° C.) for 20 minutes. The water bath washeated until the temperature in the flask reached 70° C. The reactionwas stopped after 21 hours by removing the flask from the water bath.

Table 4 lists PQ1 synthesized with addition of acid to the reactionmixture. Each sample was prepared as described above for Sample #2except with different molar ratios of reactants. No absorbance wasobserved at 228 nm, i.e., no degradation of PQ1 occurred for any of thesamples. The molecular weight was measured by the proton NMR method.

TABLE 4 Reaction PQ1 Sample Molar ratio Reaction Temper- Molecular #DA*/TEA DA/DCB*/HCl Time ature weight 1 0.83 1/1.2/0.83 5 hours 70° C.6.94k 2 1.67 1/1.1/0.41 21 hours  70° C. 11.4k 3 1.25 1/1.1/0.55 8 hours75° C.  9.3k 4 0.83 1/1.2/0.83 8 hours 60° C.  7.7k 5 1.11 1/1.15/0.62 6hours 60° C.   10k 6 5.0 1/1.1/1 18 hours  75° C.   26k *DA =1,4-bis-dimethylamino-2-butene, DCB = 1,4-dichloro-butene

As described in U.S. Pat. No. 4,027,020, PQ1 synthesis without theaddition of acid to the reaction mixture is not effective outside therange of DA/TEA molar ratio of 2:1-30:1. Table 4 above shows that themethods of the present embodiments are effective with a much largerranger of DA/TEA molar ratios. In some embodiments, PQ1 can beeffectively formed at DA/TEA molar ratio<2:1. In fact, the molecularsize of PQ1 is related to the ratio of DA/TEA: the higher the ratio, thehigher the PQ1 molecular weight.

The preferred molar ratio of the total amines (DA+TEA) to acid is fromabout 10:1 to about 1:2 and most preferably from about 5:1 to about 1:1.The preferred DA/TEA ratio is from about 0.3:1 to about 30:1 and mostpreferably from 0.8:1 to about 5:1.

The molecular weights are deduced from the proton NMR spectrum of theproduct according to the equation: mw=133.5 (6 u/v−1)+290, where u isthe peak area at the chemical shift of 6.5 ppm which is from the vinylprotons in the repeating units, and v is peak area at the chemical shiftof 3.7 ppm which is from the allylic protons adjacent to nitrogen in theending groups of the PQ1 molecules. This will be referred to as theproton

NMR method.

Example 3

An experiment was done to test the anti-bacterial effect of PQ1synthesized in the presence of acid in comparison to PQ1 moleculessynthesized without the presence of acid. Several contact lensmulti-purpose solutions were formulated by dissolving the ingredients inTable 5 in deionized water. Antimicrobial activity was tested by methodsknown in the art against the FDA contact lens disinfection panel. Logreductions at 6 hours solution contact are reported at the bottom ofTable 5.

TABLE 5 % w/w % w/w % w/w % w/w % w/w % w/w PQ1 synthesized with acidadded (sample# 5 in Table 4) PQ1 synthesized without acid * PQ-10.000075 0.0001 0.00015 0.000075 0.0001 0.00015 Hydroxypropylmethyl-0.20 0.20 0.20 0.20 0.20 0.20 cellulose (HPMC) Sodium Chloride 0.59 0.590.59 0.59 0.59 0.59 Potassium Chloride 0.14 0.14 0.14 0.14 0.14 0.14Tris HCl 0.055 0.055 0.055 0.055 0.055 0.055 Tris (base) 0.021 0.0210.021 0.021 0.021 0.021 Taurine 0.05 0.05 0.05 0.05 0.05 0.05 Poloxamer237 0.05 0.05 0.05 0.05 0.05 0.05 Edetate Disodium 0.01 0.01 0.01 0.010.01 0.01 Propylene Glycol 0.50 0.50 0.50 0.50 0.50 0.50 Purified Water98.38 98.38 98.38 98.38 98.38 98.38 Log drop at 6 hours S. marcescens13880 2.12 2.18 2.18 0.01 0.14 0.37 C. albicans 10231 0.36 0.54 0.450.21 0.21 0.17 P. aeruginosa 9027 >5.00 >5.00 >5.00 S. aureus 6538 2.892.99 3.32 F. solani 36031 2.65 2.90 3.40 * Synthesized according to theconditions described in Comparative Example 2 except the reaction timeis 40 hours.

As can be seen in Table 5, above, the antimicrobial activity is reducedconsiderably when PQ1 is generated without the presence of acid; thatis, when PQ1 is degraded. Killing 99.999% of bacteria in a sample may beexpress as a 5 log reduction. Killing 99.999% of microbes means that0.001% of the microbes survived. We started with 100% microbes and Log(100%)=0. If we have 0.001% surviving microbes, then Log (0.001%)=−5.The reduction or killing of the microbes at log scale is 0−(−5)=5. So,in the disinfecting community, 99.999% killing is called a 5 logreduction.

Comparative Example 4

PQ1 was also synthesized with the method disclosed in U.S. patentapplication Ser. No. 11/609,422, with the same amount1,4-bis-dimethylamino-2-butene, total TEA, total 6N HCl,1,4-dichloro-2-butene and water, and with the same reaction temperatureand time, as was shown in Example 4, except the second part of TEA/HClmixture was combined with the first part of TEA/HCl mixture. That is,41.2 mmoles of 1,4-bis-dimethylamino-2-butene, 16.6 mmoles of TEA, 12mmoles of HCl and 10 grams of water were combined in a 50 ml three-mouthflask. 44.8 mmoles of 1,4-dichloro-2-butene were slowly added drop-wiseinto the flask under constant stirring. After the 1,4-dichloro-2-butenewas completely added the flask was heated until the temperature insidethe flask reached 70° C. The reaction was stopped after 6 hours byremoving the flask from the water bath. The molecular weight of PQ1measured by ¹H NMR is about 14,000 Dalton.

Example 4

5.86 grams (41.2 mmoles) of 1,4-bis-dimethylamino-2-butene, 1.24 grams(8.3 mmoles) of TEA, 1 ml of 6N HCl (6 mmoles), 10 grams of water and astir bar were combined in a 50 ml three-mouth flask. 5.6 grams of (44.8mmoles) of 1,4-dichloro-2-butene were slowly added drop-wise into theflask under constant stirring. After the 1,4-dichloro-2-butene wascompletely added the flask was heated until the temperature inside theflask reached 70° C. One hour later, another mixture of 1.24 grams ofTEA and 1 ml of 6N HCl was added to the flask. The reaction was stoppedafter 6 hours by removing the flask from the water bath. The molecularweight of PQ1 measured by ¹H NMR is about 34,000 Dalton. Thus, it may beseen that adding the TEA and acid in two parts significantly increasedthe MW of the PQ1.

One of ordinary skill in the art will understand how to vary thisprocedure to scale it up for larger production. For example, variationscan be made to the procedure for larger scale the production to releasethe heat generated at the initial stage of the reaction before raisingthe temperature to above 60° C.

High Mw PQ1 as an Antimicrobial Agent in MPS

High molecular weight PQ1 has much higher antimicrobial activities thanlow molecular weight PQ1 at the same concentration level. Therefore, theeye irritation can be reduced or avoided by using high molecular weightPQ1 as a preservative/disinfecting agent for MPS or ophthalmiccompositions.

The examples below show that the activity enhancement for Sm, Fs and Apis very significant when the PQ1 average molecular weight is increasedfrom 6-7 k to about 22-34 k.

Table 6 shows antimicrobial activities for two 0.75 ppm PQ1 solutionswith NMR average molecular weight of 30,000 and 7,000 Dalton,respectively. The tests were conducted in test tube with 0.3% organicsoil added and without contact lens. The increase in activity withincrease of molecular weight is listed in the right column. As may beseen, the higher MW PQ1 had much stronger activity against S.marcescens, F. solani and C. albicans than the 6K PQ1.

TABLE 6 Log drop increase #1 #2 with PQ1 MW Ingredients % w/w % w/wincrease 30K PQ1 0.000075 7k PQ1 0.000075 Boric acid 0.60 0.60 SodiumBorate-10H20 0.18 0.18 NaCl 0.40 0.40 EDTA 0.05 0.05 Tetronic 904 0.100.10 Pluronic F87 0.05 0.05 Log drops @ 6 hours S. marcescens 13880 2.981.58 1.4 C. albicans 10231 3.43 2.77 0.66 F. solani 36031 4.17 2.45 1.72

Table 7 shows antimicrobial activities for two 1.5 ppm PQ1 solutionswith NMR average molecular weight of 30,000 and 7,000 Dalton,respectively. The tests were also conducted in test tube with 0.3%organic soil added and without contact lens. The increase in activitywith increase of molecular weight is listed in the right column. As maybe seen, the higher MW PQ1 has stronger activity against S. marcescens,F. solani and C. albicans than the 6K PQ1.

TABLE 7 Log drop increase #3 #4 with PQ1 MW Ingredients % w/w % w/wincrease 30K PQ1 0.00015 7k PQ1 0.00015 Boric acid 0.60 0.60 SodiumBorate-10H20 0.18 0.18 NaCl 0.40 0.40 EDTA 0.05 0.05 Tetronic 904 0.100.10 Pluronic F87 0.05 0.05 Log drops @ 6 hours S. marcescens 13880 3.192.56 0.63 C. albicans 10231 3.46 3.35 0.11 F. solani 36031 4.17 3.87 0.3

Table 8 shows antimicrobial activities for two 7 ppm PQ1 solutions withaverage molecular weight of 34,000 and 6,000 Dalton, respectively. Thetests were conducted in lens case with an Acuvue2 contact lens and0.003% organic soil added. The contact lenses were added while themicroorganisms were inoculated to the solution. The increase in activitywith increase of molecular weight is listed in the right column. As maybe seen, the higher MW PQ1 has stronger activity against S. marcescens,F. solani and S. aureus than the 6K PQ1.

TABLE 8 Log drop increase with PQ1 MW Ingredients % w/w % w/w increase34k PQ1 0.0007 6k PQ1 0.0007 Trisodium citrate 0.65 0.65 Boric acid 0.600.60 Sodium borate, 10 hydrate 0.125 0.125 NaCl 0.30 0.30 EDTA 0.05 0.05Tetronic 904 0.10 0.10 Pluronic F87 0.05 0.05 Log drops @ 6 hours S.marcescens 2.14 0.80 1.34 S. aureus 2.97 2.59 0.38 F. solani 3.63 1.931.70

Table 9 shows antimicrobial activities for two 10 ppm PQ1 solutions withaverage molecular weight of 34,000 and 6,000 Dalton, respectively. Thetests were conducted in lens case with an Acuvue2 contact lens and0.003% organic soil added. The contact lenses were added while themicroorganisms were inoculated to the solution. The increase in activitywith increase of molecular weight is listed in the right column. As maybe seen, the higher MW PQ1 had much stronger activity against S.marcescens, F. solani and S. aureus than the 6K PQ1.

TABLE 9 Log drop increase with PQ1 MW Ingredients % w/w % w/w Increase34k PQ1 0.001 6k PQ1 0.001 Trisodium citrate 0.65 0.65 Boric acid 0.600.60 Sodium borate, 10 hydrate 0.125 0.125 NaCl 0.30 0.30 EDTA 0.05 0.05Tetronic 904 0.10 0.10 Pluronic F87 0.05 0.05 Log drops @ 6 hours S.marcescens 2.07 0.88 1.19 S. aureus 3.12 2.78 0.34 F. solani 4.18 1.952.23

Table 10 shows anti-Acanthamoeba activities for 7 and 10 ppm PQ1solutions with average molecular weight of 34,000 and 6,000 Dalton,respectively. The tests were conducted in test tube with 0.3% organicsoil added. As may be seen, for both concentrations tested, theanti-Acanthamoeba activity is significantly higher with the highermolecular weight PQ-1.

TABLE 10 Ingredients % w/w % w/w % w/w % w/w 34k PQ1 0.0007 0.0010 6kPQ1 0.0007 0.0010 Trisodium citrate 0.65 0.65 0.65 0.65 Boric acid 0.600.60 0.60 0.60 Sodium borate, 10 hydrate 0.125 0.125 0.125 0.125 NaCl0.30 0.30 0.30 0.30 EDTA 0.05 0.05 0.05 0.05 Tetronic 904 0.10 0.10 0.100.10 Pluronic F87 0.05 0.05 0.05 0.05 Log drops @ 6 hours A polyphage1.09 1.36 0.14 0.88

Table 11 shows antimicrobial activities for 7 ppm PQ1 solutions withaverage molecular weight of 28,300, 22,900 and 6,000 Dalton,respectively. The tests were conducted in lens case with Acuvue2 contactlenses and 0.003% organic soil added. The microorganisms were inoculated40 hours after the contact lenses were added to the solution. As may beseen, the 28.3 k and 22.9 k PQ1 perform similarly. Further, both of thehigher MW PQ1s have much stronger activity against S. macescens, F.solani and Acanthemoeba than the 6 k PQ1.

TABLE 11 Ingredients % w/w % w/w % w/w 28.3k PQ1 0.0007 22.9k PQ1 0.00076k PQ1 0.0007 Trisodium citrate 0.65 0.65 0.65 Boric acid 0.60 0.60 0.60Sodium borate, 10 hydrate 0.125 0.125 0.125 NaCl 0.30 0.30 0.30 EDTA0.05 0.05 0.05 Tetronic 904 0.10 0.10 0.10 Pluronic F87 0.05 0.05 0.05Log Drops @ 6 hours S. marcescens 13880 2.01 2.03 1.26 S. aureus 65382.77 2.45 2.37 F. solani 36031 >4.52 >4.52 2.22 A. polyphaga 30461 1.671.49 0.75

Table 12 shows antimicrobial activities for 10 ppm PQ1 solutions withaverage molecular weight of 28,300, 22,900 and 6,000 Dalton,respectively. The tests were conducted in lens case with Acuvue2 contactlenses and 0.003% organic soil added. The microorganisms were inoculated40 hours after the contact lenses were added to the solution. As may beseen, the 28.3 k and 22.9 k PQ1 clearly outperform the 6 k PQ-1.Further, both of the higher MW PQ1s have much stronger activity againstS. macescens, F. solani and Acanthamoeba than 6 k PQ1.

TABLE 12 Ingredients % w/w % w/w % w/w 28.3k PQ1 0.001 22.9k PQ1 0.0016k PQ1 0.001 Trisodium citrate 0.65 0.65 0.65 Boric acid 0.60 0.60 0.60Sodium borate, 10 hydrate 0.125 0.125 0.125 NaCl 0.30 0.30 0.30 EDTA0.05 0.05 0.05 Tetronic 904 0.10 0.10 0.10 Pluronic F87 0.05 0.05 0.05Log Drops @ 6 hours S. marcescens 13880 2.19 2.21 1.26 S. aureus 65383.03 2.64 2.38 F. solani 36031 >4.52 >4.52 2.81 A. polyphaga 30461 1.731.77 0.81

As demonstrated above, higher molecular weight PQ1 has much higherantimicrobial activities than low molecular weight PQ1 at the sameconcentration level. This is especially true when a contact lens ispresent during disinfection. To reach the same disinfection efficacy asthat of high molecular weight PQ1, the low molecular weight PQ1solutions have to have significantly higher PQ1 concentrations. A higherPQ1 concentration generally results in more PQ1 lens uptake. Thisincrease in lens uptake generally results in a greater PQ1 release inthe eye, and increased eye irritation. Therefore, the eye irritation canto reduced or avoided by using high molecular weight PQ1 as apreservative/disinfecting agent for MPS or ophthalmic compositions.Table 13 shows two different ophthalmic formulations, both of whichcontain the same concentration of PQ-1.

TABLE 13 1 2 % w/w % w/w Alexidine 0.00018 7k PQ1 0.00018 30k PQ-10.00018 Boric Acid 0.6 0.6 Sodium Borate 10H20 0.17 0.17 NaCl 0.4 0.4EDTA 0.05 0.05 Tetronic 904 0.1 0.1 Pluronic F87 0.05 0.05 HA 0.00250.0025 cytotoxicity score 2 3

A direct overlay method which has been adopted by the FDA for MPSregistration was used to evaluate the cytotoxicity profile of the PQ1formulations. In this method, previously-soaked contact lenses (in 100ml of the solutions to simulate overnight soak) were directly overlayedon L929 cells. The cytotoxicity result was evaluated by scoring eachculture under a microscope on a relative scale of 0-4. A score of 2indicates that cell damage is limited to the area under the lens. Ascore of 3 indicates that the cell damage extends 0.5 to 1.0 cm beyondthe lens. Therefore, this shows that the high molecular weight PQ1solution has a lower cytotoxicity than the low molecular weight PQ1solution.

One possible explanation of the lower cytotoxicity is that the large PQ1molecules are more difficult to get into the hydrogel lens matrixmaterial do to the limited pore size of the lens material itself. Asnoted above, the lower the lens uptake of PQ1, the less PQ1 will bereleased to the cells. The reduced amount of high molecular weight PQ1loss from the solution to the lens is consistent with the observationabove that high molecular weight PQ1 is more efficacious againstmicrobes in the presence of contact lenses.

Equivalents

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription details certain preferred embodiments of the invention anddescribes the best mode contemplated by the inventor. It will beappreciated, however, that no matter how detailed the foregoing mayappear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

1. A method of making antimicrobial quaternary ammonium polymers,comprising: a) mixing 1,4-bis-dimethylamino-2-butene, water, a firstportion of triethanolamine and a first portion of acid; b) adding a1,4-dichloro-2-butene and heating the reaction mixture; c) adding asecond portion of triethanolamine and a second portion of acid, and d)isolating a quaternary ammonium polymer having a molecular weightdetermined using the proton NMR method of at least 26,000.
 2. The methodof claim 1, wherein said quaternary ammonium polymers includepolyquaternium-1 having a molecular weight of at least 28,000.
 3. Themethod of claim 1, wherein the acid is selected from the groupconsisting of HCl, H₂SO₄ and H₃PO₄.
 4. The method of claim 1, whereinthe acid is HCl.
 5. The method of claim 1, wherein the1,4-dichloro-2-butene is added drop-wise.
 6. The method of claim 1,wherein the molar ratio of 1,4-bis-dimethylamino-2-butene totriethanolamine is from about 10:1 to about 1:5.
 7. The method of claim1, wherein the molar ratio of triethanolamine to acid in said firstportion is from about 10:1 to about 1:10.
 8. The method of claim 1,wherein the molar ratio of triethanolamine to acid in said secondportion is from about 10:1 to about 1:100.
 9. The method of claim 1,wherein the molar ratio of triethanolamine to acid in said first andsecond portions is from about 5:1 to about 1:5.
 10. The method of claim1, wherein the reaction temperature is from about 10° C. to about 90° C.11. The method of claim 1, wherein the reaction time is from about 1hour to about 40 hours.
 12. A method of making Polyquaternium-1,comprising: a) mixing 1,4-bis-dimethylamino-2-butene, water, a firstportion of triethanolamine and a first portion of acid; b) introducing a1,4-dichloro-2-butene and heating the reaction mixture; c) adding asecond portion of triethanolamine and a second portion of acid, and d)isolating Polyquaternium-1 having a molecular weight determined usingthe proton NMR method of 26,000 or more, at a yield of at least about50%.
 13. The method of claim 12, wherein said quaternary ammoniumpolymers include polyquaternium-1 having a molecular weight of at least28,000.
 14. The method of claim 12, wherein the acid is selected fromthe group consisting of HCl, H₂SO₄ and H₃PO₄.
 15. The method of claim12, wherein the acid is HCl.
 16. The method of claim 12, wherein the1,4-dichloro-2-butene is added drop-wise.
 17. The method of claim 12,wherein the molar ratio of 1,4-bis-dimethylamino-2-butene totriethanolamine is from about 10:1 to about 1:5.
 18. The method of claim12, wherein the molar ratio of triethanolamine to acid in said firstportion is from about 10:1 to about 1:10.
 19. The method of claim 12,wherein the molar ratio of triethanolamine to acid in said secondportion is from about 10:1 to about 1:100.
 20. The method of claim 12,wherein the molar ratio of triethanolamine to acid in said first andsecond portions is from about 5:1 to about 1:5.
 21. The method of claim12, wherein the reaction temperature is from about 10° C. to about 90°C.
 22. The method of claim 12, wherein the reaction time is from about 1hour to about 40 hours.